The medical proteins or industrial enzymes useful for humans, which could only be obtained in a trace amount from the natural state in the past, could be mass-produced by the development of recombinant DNA technology. For example, E. coli cells have been most widely used as host cells to produce large amounts of such useful proteins, and useful recombinant proteins, including hormones such as insulin and β-endorphin, and immunomodulators such as interferon, have been researched and developed.
To efficiently produce recombinant proteins, selection of suitable host cells is very important. As host cells for producing therapeutic recombinant proteins, various host systems, including microbial, plant and animal cells, have been developed and used. Particularly, for most glycoproteins, animal cells that are higher eukaryotic cells have been used as host cells. However, animal cells have shortcomings in that they are cultured using expensive media, show low protein production yields, and are cultured under strict conditions. For this reason, for non-glycoproteins, microorganisms are used as hosts.
Among various microbial host systems, E. coli and yeast are mainly used as primary host cells for producing large amounts of recombinant proteins. These microbial expression systems have advantages over higher eukaryotic cell expression systems in that the production cost is low and the production process is simple. However, there is a limit to the production of either glycoproteins that require post-translational modification such as glycosylation to have activity, or proteins having a very large and complex structure. Furthermore, when a useful protein is expressed in yeast, an insoluble inclusion body protein is formed which lost its activity by various mechanisms without being completely folded. Although this insoluble protein may be easily isolated in an initial stage to provide a highly pure protein in some cases, it lacks activity as the protein. For this reason, complex and costly denaturation and refolding processes are required to obtain a biologically active soluble protein from the insoluble protein.
Furthermore, even if cell lines for producing recombinant protein drugs are established, studies on processes for production of recombinant proteins are required to identify quality and characteristics for cell lines, and the development of scale-up production processes is also required. The protein production processes are largely divided into an upstream process of establishing a host cell line, a midstream process of culturing the cell line to produce a large amount of recombinant protein, a downstream process for separation and purification, and a process of formulating a purified drug substance with an excipient or the like. For each of such unit processes, optimal conditions for key process parameters need to be established, thereby establishing optimal production process conditions.
Meanwhile, interleukin-2 consists of 153 amino acids and is produced mainly by T cells expressing the surface antigen CD4. Transformed T cells, B cells, lymphocytic cancer cells, LAK cells and NK cells also secrete interleukin-2. It is known that the production of interleukin-2 is induced by mitogen- or allergen-mediated activation of T cells, and several kinds of secondary stimulations are required to maximize the production of interleukin-2, but resting cells cannot produce interleukin-2. It has been reported that interleukin-2 and its receptor are associated with many diseases. However, studies on the molecular characteristics of interleukin-2 and its receptor have been very limited, because they are obtained in limited amounts.
For example, many methods have been studied to increase immunity against cancer by administration of functional interleukin-2 gene, and thus studies on interleukin-2 and the demand for interleukin-2 as a therapeutic agent have continued to increase. However, technology for producing a large amount of interleukin-2 is still insufficient.
Under this background, there is a need for studies on an optimized method for producing a large amount of interleukin-2 using a microbial expression system.